Pulmonary surfactant is known to be critical for gas exchange and for maintaining patency of small airways. However, regulation of surfactant production in the adult lung is not completely understood. There is currently no known pharmacologic means to increase endogenous surfactant production in vivo for the treatment of lung diseases. We recently revisited the use of air/liquid interface cultures (no apical fluid) in the primary culture of type II cells. We found that the addition of apical fluid rapidly (<48 h) reduces surfactant protein levels and removal of all apical fluid rapidly (<48 h) restores surfactant protein levels. The purpose of this R21 grant is to discover the mechanism for this effect. We propose that the mechanism is oxygen sensing by prolyl hydroxylase 2 (PHD2) and that the effect is HIF1a mediated. We find that in submerged cultures there is an increase in nuclear HIF1a and HIF2, an increase in HIF responsive genes, the effect is reproduced by the HIF stabilizer, DMOG, and that the effect is blocked by supplemental oxygen. The downstream alterations likely include inhibiting two key transcription factors C/EBPa and SREBP-1. However, we are mindful that other pathways are also likely involved, and we will examine TTF1, FOXA2, mTOR, PPAR? and AMPK target genes in the analyses of the microarray experiments. Our initial focus will be on HIF1a, C/EBP1, and SREBP-1c responsive genes, since they are likely to be involved. We have significantly improved our type II cell cultures so all the studies in this proposal will be human type II cells. We will address both surfactant protein and phospholipid synthesis. We believe that the deacylation-reacylation remodeling pathway for dipalmitoylphosphatidylchoine (DPPC) is less active in human cells than in rodent cells based on the regulation of gene expression of lysophosphatidylcholine acyltransferase (LPCAT1). The goal of this study is to define the regulation of surfactant production in adult human type II cells. This study should also indicate that sustained pulmonary edema will impair type I cell function and provide another reason for the use of supplemental oxygen in acute lung injury.